title: Measurement of blood protease kinetic parameters with self-assembled monolayer ligand binding assays and label-free MALDI-TOF MS.
abstract: We report novel ligand binding assay (LBA) surface modalities that permit plasma protease catalytic efficiency (kcat/km) determination by MALDI-TOF MS without the use of liquid chromatography or internal standards such as chemical or metalized labels. Two model LBAs were constructed on planar self-assembled monolayers (SAMs) and used to evaluate the clinically relevant metalloprotease ADAMTS-13 kinetics in plasma. The SAM chemistries were designed to improve biosampling efficiency by minimization of nonspecific adsorption of abundant proteins present at ~100,000× the concentration of the endogenous enzyme. In the first protocol, in-solution digestion of the ADAMTS-13 substrate (vWFh) was performed with immunoaffinity enrichment of the reaction substrate and product to SAM arrays. The second configuration examined protease kcat/km via a surface digestion modality where different substrates were covalently immobilized to the SAM at controlled surface density for optimized protease screens. The results show the MALDI-TOF MS LBA platforms provide limits of quantitation to ~1% protease activity (~60 pM enzyme concentration) in <1 h analysis time, a ~16× improvement over other MS-based LBA formats. Implementation of a vacuum-sublimed MALDI matrix provided good MALDI-TOF MS intra- and interday repeatability, ~1.2 and ~6.6% RSD, respectively. Platform reliability permitted kcat/km determination without internal standards with observed values ~10× improved versus conventional fluorophoric assays. Application of the assays to 12 clinical plasma samples demonstrated proof-of-concept for clinical applications. Overall, this work demonstrates that rationally designed surface chemistries for MALDI-TOF MS may serve as an alternative, label-free methodology with potential for a wide range of biotechnology applications related to targeted enzyme molecular diagnostics.
authors.affiliation: Department of Pathology, University of Texas Southwestern Medical Center , Dallas, TX 75390-9072.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Patrie
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Roth
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Plymire
authors.forename: Erica
authors.initials: E
authors.lastname: Maresh
authors.forename: Junmei
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000097:blood* / Q000737:chemistry; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D008024:Ligands; D010447:Peptide Hydrolases